Mirza S Baig1, Usman Yaqoob2, Sheng Cao2, Uzma Saqib3, Vijay H Shah4. 1. Center for Biosciences and Biomedical Engineering (BSBE), Indian Institute of Technology Indore (IITI), Indore, MP, India; Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. Electronic address: msb@iiti.ac.in. 2. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. 3. Discipline of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore (IITI), Indore, MP, India. 4. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. Electronic address: Shah.Vijay@mayo.edu.
Abstract
AIMS: Matrix metalloproteinases (MMPs) that degrade extracellular matrix (ECM) and help to resolve the excess matrix are considered to be under-expressed in fibrosis. MMPs are generally anti-fibrotic, however others can have pro-fibrotic functions. Therefore, the aim of this study was to find out the mechanism of pro-fibrotic function of MMPs in hepatic stellate cells' (HSC's) activation and migration. MAIN METHODS: Human MMP Antibody Array from Abcam was used to profile MMPs in macrophages. Gelatin or casein zymography was performed using 10% SDS-polyacrylamide gels (SDS-PAGE) containing gelatin (1mg/ml) or Casein (1mg/ml) as substrate. HSCs migration assay was performed using Boyden chamber as described previously (Guo et al., 2007, McGarrigle et al., 2006, Shan et al., 2006 and Yang and Huang, 2005). Real-time PCR with SYBR green was performed using iTaq™ universal SYBR® Green supermix (BIO-RAD) and a 7500 Real-Time PCR System (Applied Biosystems). Collagen, type I, alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA) expression was determined by immunoblot analysis. KEY FINDINGS: We first profiled the expression of all MMPs in primary murine bone marrow-derived macrophages (BMDMs) and differentiated THP-1 cells and found that MMP-8, -10, & -13, were significantly overexpressed after 12h of lipopolysaccharide (LPS) treatment. Based on this pattern of expression, we speculated that macrophage MMP-8,-10, &-13 might play a non-canonical role in HSCs activation. Further, we found that exogenous active MMP-8 (Collagenase-2) treated HSC shows markedly increased migration and COL1A1 expression as compared to MMP-10 and MMP-13 treated HSCs. Thus, macrophage MMP-8 (Collagenase-2) expression in macrophages emerges as an important moderator of HSC cell migration and invasion. SIGNIFICANCE: These findings suggest that macrophage MMP-8 promotes HSC activation and might have a role in liver disease progression. MMP-8 targeting in the liver may have therapeutic potential in alcoholic liver disease (ALD).
AIMS: Matrix metalloproteinases (MMPs) that degrade extracellular matrix (ECM) and help to resolve the excess matrix are considered to be under-expressed in fibrosis. MMPs are generally anti-fibrotic, however others can have pro-fibrotic functions. Therefore, the aim of this study was to find out the mechanism of pro-fibrotic function of MMPs in hepatic stellate cells' (HSC's) activation and migration. MAIN METHODS:Human MMP Antibody Array from Abcam was used to profile MMPs in macrophages. Gelatin or casein zymography was performed using 10% SDS-polyacrylamide gels (SDS-PAGE) containing gelatin (1mg/ml) or Casein (1mg/ml) as substrate. HSCs migration assay was performed using Boyden chamber as described previously (Guo et al., 2007, McGarrigle et al., 2006, Shan et al., 2006 and Yang and Huang, 2005). Real-time PCR with SYBR green was performed using iTaq™ universal SYBR® Green supermix (BIO-RAD) and a 7500 Real-Time PCR System (Applied Biosystems). Collagen, type I, alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA) expression was determined by immunoblot analysis. KEY FINDINGS: We first profiled the expression of all MMPs in primary murine bone marrow-derived macrophages (BMDMs) and differentiated THP-1 cells and found that MMP-8, -10, & -13, were significantly overexpressed after 12h of lipopolysaccharide (LPS) treatment. Based on this pattern of expression, we speculated that macrophage MMP-8,-10, &-13 might play a non-canonical role in HSCs activation. Further, we found that exogenous active MMP-8 (Collagenase-2) treated HSC shows markedly increased migration and COL1A1 expression as compared to MMP-10 and MMP-13 treated HSCs. Thus, macrophage MMP-8 (Collagenase-2) expression in macrophages emerges as an important moderator of HSC cell migration and invasion. SIGNIFICANCE: These findings suggest that macrophage MMP-8 promotes HSC activation and might have a role in liver disease progression. MMP-8 targeting in the liver may have therapeutic potential in alcoholic liver disease (ALD).